Many research programs use well-established tumor cell lines as tumor models for in vitro drug screening studies. Because the tumor spheroids, tumor cells grown as three-dimensional (3-D) structures are multicellular more like the tumors in vivo than monolayer culture cells, use of tumor cell spheroids as in vitro models for drug development is of great interest. Monotypic spheroids, however, do not model the stromal-epithelial interactions that have an important role in controlling tumor growth and development in vivo.
The tissue engineering lab at Huntington Medical Research Institutes (HMRI) in Pasadena, Calif. under the direction of Dr. Marylou Ingram discovered a method for generating, reproducibly, more realistic 3-D tumor models that contain both stromal and malignant epithelial cells with an architecture that closely resembles tumor micro-lesions in vivo. Because they are so tissue-like they are referred to as tumor histoids. The bioreactor developed to generate histoid constructs is described and illustrated in U.S. Pat. No. 6,998,264, the contents of which are incorporated herein by reference. Examples of histological sections of tumor histoids representing cancers of breast, prostate, colon, pancreas and urinary bladder are described.
The 3D heterocellular tumor histoids, produced with no scaffolds or extracellular matrix addition, allow the cells to contact each other in a way very similar to cells in natural tumors. These contacts enable development of a stromal microenvironment that closely approximates the in vivo environment of natural tumors. Therefore 3D tumor histoids potentially provide a significant advantage over 2D monolayer cultures typically used for studies of cancer biology and for pre-clinical drug screening, testing and development. However such multicellular 3D models are currently used as drug testing platforms without thorough molecular characterization that would be advantageous for evaluation of their use for modeling of in vivo treatment conditions in place of 2D monolayer cultures. In addition it is not known whether the in vitro 3D models produced by different methods, for example by low shear microgravity or high throughput hanging drop, are molecularly similar or identical. The present invention is based at least in part on the recognition that this information would be useful for eliminating variability in the use of these models for drug testing. Furthermore, the inventor recognized that delineating the molecular characteristics unique to 3D cultures could yield both diagnostic and therapeutic targets that are superior to those identified through molecular characterization of 2D monolayer cultures as compared to healthy cells. Thus molecular characterization of the features that uniquely distinguish these 3D in vitro model products has heretofore constituted an unmet need.
In recent years the mature small RNAs, termed the microRNAs (miRNAs) have emerged as important biological regulatory molecules playing a pivotal role in gene expression of plants and animals, including humans. miRNAs are integral part of miRNA-protein RISC complex that regulates messenger RNAs (mRNAs) at translational level through binding target mRNAs at their 3′ untranslated region (3′UTR). A single miRNA can regulate expression of multiple genes and thus act as a global modulator of diverse cellular and biological processes. Moreover miRNA expression profile can be indicative of physiological state of a cell. For example the expression profiles of a few hundred miRNA have provided more specific classification of human cancers than mRNA profiles (Lu, J., et al., Nature, 2005, 435(7043), 834-838). Unique expression profiles identified in lung cancers position miRNA as diagnostic and prognostic markers (Yanaihara, N., et al., Cancer Cell, 2006, 9(3), 189-198).
The prior art studies however did not attempt to elucidate the biological significance of changes in miRNA expression profile in vitro tumor model tissues.
The present invention is based at least in part on the recognition that such a study would allow for identification of cellular pathways whose dysregulation underlies tumor development and maintenance. The inventor also recognized that this kind of analysis would be useful in characterizing the differences between 3D heterocellular tumor histoids and 2D monolayer cultures of cancer cells, since it would allow for delineation of the molecular features that are unique to the biology of 3D culture micro-tissues, and that are more likely to be present in natural tumors. The inventor further recognized that pathways so identified could provide superior diagnostic and therapeutic candidates as compared with pathways identified in comparison of healthy cells and 2D monolayer cultures of cancer cells. Moreover, the inventor recognized that, if a given signaling pathway is found to be dysregulated in a particular tumor, knowing its status as unique to 3D cultures or common to both 3D and 2D cultures would allow for an informed decision as to which system to use as the drug development platform. Since using 3D cultures is significantly more costly, an informed choice could result in significant savings in cost and possibly time. Thus, the inventor recognized that proper characterization of miRNAs and cellular pathways unique to 3D heterocellular tumor histoid could go a long way to satisfying the need described above.